The possible role of telomeres in the short life span of the bay scallop, Argopecten irradians irradians (Lamarck 1819).

ABSTRACT The life spans of eukaryotes can be determinate or indeterminate. Examples of the latter are found from sponges to lobsters to rainbow trout that reproduce and grow until disease, predation, or environmental circumstances end their lives. Their tissues continually express the enzyme telomerase, keeping sufficient telomeres on the end of their chromosomes to avoid individual cell senescence and subsequent death. Bay scallops, on the other hand, have a well-defined life span, generally between 18-22 mo in the northeastern USA, and reproduce usually once. Argopecten irradians irradians (L.) has been found to possess fewer telomeres than a close relative, Argopecten purpuratus (L.), a cold water species found along the coasts of Peru and Chile that can live to 7 y or more. A. purpuratus contains significantly more telomeres than A. irradians in their respective tissues. It is proposed that the short lifespan of A. irradians may not be a selective advantage, but rather because of an evolutionary loss of telomeres through Robertsonian fusion and extensive chromosome arm loss. Evidence for this is seen by the bay scallop doubling its weight postspawning and storing nutrients in the form of carbohydrates, lipids, and protein for the upcoming winter and the subsequent initiation of gametogenesis in the spring. However, they rarely survive to complete a second reproductive event. This life history suggests that A. irradians is not a semelparous species (one-time reproduction) but rather an example of interrupted iteroparity (repeated reproduction) caused by losses in its genome. Adding telomeres to the ends of chromosomes of A. irradians may extend the life span of this species, possibly permitting several reproductive seasons ahead. In years when larval recruitment failure occurs without concomitant loss of adult spawners, local populations could recover in as little as a single season.

KEY WORDS: telomeres, bay scallop, Argopecten, aging, life span

INTRODUCTION

Several theories of aging have been developed over the years. Pearl (1928; cited in Carlson & Riley 1998) saw an inverse relationship between longevity and metabolism, with the harmful results of an excessive metabolic rate leading to an earlier demise. The free radical theory, put forth by Harman (1956), states that highly reactive products of metabolism such as reactive oxygen species (ROS) can be damaging to tissues. Another theory assumes the constant onslaught of environmental insults (e.g., solar radiation, toxic effects of ingested materials, and the like) cause DNA damage that outstrips the body's ability to repair it (Carlson & Riley 1998). A fourth suggests that genes producing favorable characteristics in early life may become harmful after reproduction (Rose 1991). Recent progress in the field of molecular biology has led to new methods of studying aging in eukaryotes. Telomeres, nucleotide repeats found at the end of chromosomes, have been shown to act as a "mitotic" clock that may define the life span of a species (Blackburn 1991, Harley 1991, Wright & Shay 2005). With each round of cell replication, a number of telomeres are lost (Blackburn 1991, Levy et al. 1992).

Senescence may occur when telomere length reaches a critical length, inducing changes that resemble DNA breaks and subsequent checkpoint arrest (Zou et al. 2002). Although the exact connection between telomere loss and cell senescence has not been elucidated, it is known that the cell cycle control protein, p53, is located near telomeres (Levine et al. 1993), and other studies have shown a relationship between telomere loss and aging (Kulju & Lehman 1995, Vaziri & Benchimol 1996, Whikehart et al. 2000). There have been some studies that correlate longer telomeres with longer survival. Experiments have shown that reconstituting active telomerase in cells yields elongated telomeres and an extended life span in human tissue culture (Bodnar et al. 1998, Vaziri & Benchimol 1998). Joeng et al. (2004), overexpressed the telomere-binding protein, HRP-1 in the worm, Caenorhabditis elegans, resulting in longer telomeres and subsequent longer life. Tree swallows (Tachycineta bicolor) with longer telomeres have been shown to live longer than those with shorter telomeres (Haussmann et al. 2005). Conversely, experiments that prevented telomerase from adding more telomeres to the ends of chromosomes resulted in telomere shortening and cell death (Herbert et al. 1999).

Aging in many invertebrates and some vertebrates does not exist (i.e., these animals simply continue to grow and reproduce until environmental conditions, disease or predation finally end their lives). Klapper et al. (1998a) discovered the enzyme telomerase in all tissues of the lobster, Homarus americanus. Similarly, Koziol et al. (1998) found high levels of telomerase in the tissues of sponges studied. The rainbow trout, Oncorhynchus mykiss is considered "immortal" because of its similar telomerase distribution (Klapper et al. 1998b). Normally, species with a defined life span do not exhibit telomerase activity in their somatic cells, and the subsequent telomere loss determines the length of their lives (Harley et al. 1992, Shay & Wright 2000). The ages of survival of some commercial bivalve molluscs range from less than two years in the genus Argopecten, (Belding 1910) to the deep-ocean clam, Artica islandica, living specimens of which have been aged at more than 150 y (Thompson et al. 1980, Ropes 1985). Another long-lived deep-sea bivalve, Tindaria callistifomis, attains an average length of 8.4 mm after 100 y and is sexually mature after 50 60 y (Turekian et al. 1975). It is currently unknown whether these molluscs have telomerase in all of their tissues as is seen in the lobster, or have a much lower cell turnover because of their deep-sea environment coupled with a sufficient complement of telomeres. Research has shown that representative molluscs demonstrate the same telomeric repeat, [(TTAGGG).sub.n] as is found in all vertebrates studied to date (Meyne et al. 1989). For example, the oyster. Crassostrea gigas, (Guo & Allen 1997), the bay scallop, Argopecten irradians irradians (Estabrooks 1999), the wedgeshell clam, Donax trunculus, (Plohl et al. 2002), and the land snails, Cantareus aspersus and C. mazzullii, (Vitturi et al. 2005), all share this same sequence. However, there have been no studies to date, that demonstrate a correlation of age and senescence with telomere length in the Mollusca. Epp et al. (1988) looked for possible causes of senescence in the bay scallop, A. irradians and postulated that there might be a connection between protein metabolism and neurosecretory cycles resulting in a possible neuroendocrine disturbance. Barber and Blake (1986), speculated that the high cost of reproduction may accelerate the process of senescence in the southern subspecies, A. irradians concentrieus. Bricelj et al. (1987b) concluded that senescence in the second year bay scallop was not linked to the metabolic costs of an upcoming second reproductive effort.

The current study compared the telomeres of two closely related species of Argopecten. namely A. purpuratus, capable of surviving 7 10 y or more (DiSalvo et al. 1984, Alarcon & Wolff 1991), and A. irradians irradians, with a life span less than 2 y. The genus, Argopecten, split off around 19 million years ago during the middle Miocene (Waller 1969), whereas Argopecten purpuratus, found off the coasts of Peru and Chile, arose approximately 6 million years ago and became separated from the Atlantic stock with the closing of the Atlantic-Pacific connection at the end of the Miocene, whereas Argopecten irradians split off around 1.5 million years ago during the Pleistocene when rising water levels produced the bays and sounds of today (Waller 1969). After first confirming that the telomeric sequence of A. purpuratus was the same as that for A. irradians [(TTAGGG).sub.n] their telomere lengths were compared as a possible explanation for the difference in their respective life spans. In addition, the subtelomeric sequences of A. irradians were compared with those of Placopecten magellanicus, a species with the modal number of n = 19, that arose some 65 million years ago. It has been shown that the more recent the species, the more numerous the subtelomeric patches that are sites of high rates of interchromosomal recombination and with both positive, and negative outcomes (Rocco et al. 2001, Linardopoulou et al. 2005).

An argument is presented for the possible benefits of extending the life span of Argopecten irradians through telomere elongation as an additional approach to population recovery and maintenance of adequate stocking densities in this species.

METHODS AND MATERIALS

Three to four bay scallops belonging to each cohort of A. irradians were collected at approximately six-month intervals from the same locale in Nantucket Harbor, MA. This ensured that all comparisons of telomere lengths were of scallops approximately one year apart in age. Specimens of Argopecten purpuratus were obtained through the generosity of Karin Lohrmann Sheffield from the Universidad Catolica del Norte in Chile and Louis DiSalvo from Chile. DNA was extracted from the digestive gland, kidney, adductor muscle, heart and gill tissues either by the method using DNAzol (Molecular Research, Inc., Cincinnati, OH), see Estabrooks (1999). or a slight modification of the method of Sokolov (2000) that yields large amounts of DNA free of mucopolysaccharides often found in molluscan tissue. In the latter method, small amounts of tissue samples, 50-70 [micro]g in 1.5-mL tubes, were homogenized briefly in 1.0 mL of lysing reagent (50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 1% sodium dodecyl sulphate (SDS), 0.2-0.4 mg/mL proteinase K) and incubated for 2 h at 55[degrees]C. 100 [micro]l of saturated KCl were added, mixed and the tubes placed on ice for 5 min. After spinning at x 14,000g, the supernate was transferred to new tubes and extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) using the Eppendorpf Phase-Lok system (Westbury, NY). The supernate was then transferred to a new tube and the DNA precipitated with an equal amount of 100% ethanol, washed twice in 70% ethanol, drained and reconstituted with TE buffer, pH 8.0. RNase A was added at 0.2 mg/ml and the DNA was then quantified at 260/280 nm with a Beckman DU7 spectrophotometer. Known amounts of DNA were cut with the restriction enzymes RsaI (Sigma-Aldrich, St. Louis, MO) and HinfI (Amersham Biosciences, Piscataway, NJ) according to manufacturers recommendations and electrophoresed on 0.8% agarose gels for approximately one hour at 105V. Each electrophoretic run consisted of DNA from year 1 and year 2 bay scallops taken at different times from Nantucket Harbor. Scallops in each age group were from the same area of the Harbor and hence from the same cohort, helping to ensure a 12 mo difference in age at each sampling time. The DNA was then transferred to nylon membranes by Southern blotting in 0.4M NaOH, rinsed briefly in 2x SSC and air-dried between sheets of 3MM filter paper. The membrane was then baked at 80[degrees]C for 30 min to fix the DNA to the membrane. Telomeres were detected using the chemiluminescent procedure described in Estabrooks (1999). Average telomere lengths were estimated using molecular weight markers (kb) included in each run.

RESULTS

Telomeres are expressed as telomere restriction fragments (TRF) because they are cut from the ends of the chromosome by restriction enzymes at a point below the innermost telomeric (TTAGGG) segment and may contain a small amount of nontelomeric DNA.

Figure 1 follows the decline in the telomere lengths of the digestive gland of Argopecten irradians as compared that of A. purpuratus. A year 1 A. irradians is from 0-12 mo at which point it is generally sexually mature and ready to spawn. Semiquantatative measurements determined that the average telomere lengths for A. purpuratus (2-y-old) to be approximately 9.4 kb. A representative telomere run for A. irradians at approximately 6 mo old yielded a length of 2.3 kb, and 0.75 kb for A. irradians at 18 mo. Samples of year 1 and year 2 cohorts of A. irradians were collected together from Nantucket harbor. This ensured that all comparisons of telomere lengths were of scallops approximately one year apart in age and all demonstrated a large drop in telomere lengths between the two cohorts. At no time did a year 2 scallop have telomeres longer than those of any year 1, and only the digestive gland tissue demonstrated the dramatic differences in length, most likely because of a much higher rate of cellular turnover.

[FIGURE 1 OMITTED]

A year 2 scallop is from 13 24 mo, but will generally not survive to complete a second year. The TRFs of a bay scallop entering its 3rd year (24 mo plus) are shown in Figure 2, comparing those of its cardiac tissue with those of the digestive gland. The heart muscle does not divide, and so demonstrates a uniform group of long (high MW) telomeres that do not shorten with age, whereas the digestive gland has very few telomeres of any length. The level of telomeres in the adductor muscles of two cohorts of A. irradians also remains the same as would be expected, as muscle tissue generally does not replicate (Fig. 3).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Figure 4 compares the digestive gland telomeres of the long-lived deep-sea scallop, Placopecten magellanicus (4-y-old) with those of a year 1 bay scallop. Note the scarcity of subtelomeric sequences in P. magellanicus, giving support to the theory that the more recent the species, the more subtelomeric sequences may be found (Meyne et al. 1990, Rocco et al. 2001).

[FIGURE 4 OMITTED]

DISCUSSION

The chromosome number of the extant species of scallops has the modal haploid number, n = 19, whereas the bay scallops, A. irradians and A. purpuratus have n = 16 (Wada 1978, Gajardo et al. 2002). This reduction in chromosome number may be because of Robertsonian fusion, wherein telocentric chromosomes fused, reducing the total chromosome number (Wang & Guo 2004). They also noted that A. irradians incurred chromosome arm losses of nearly 50% as compared with the modal number 2 n = 38, and suggested that an ancestral bivalve may have become tetraploid at some point in time as an explanation of how Argopecten was able to sustain such extensive chromosome losses and still survive.

Most of the scallop species studied to date have the modal chromosome number n = 19, and live longer than the bay scallop (Beaumont & Zouros 1991). It is suggested that key DNA material involved in longevity may have been lost in some scallop species along the way, leaving A. irradians with just enough to survive a single reproductive effort.

As further evidence, Rocco et al. (2001) demonstrated an increasing number of subtelomeric sequences of TTAGGG in several Chondrichthian species at different stages of evolution that resulted in chromosome reduction. This may be the case with the bay scallop. The more recent the species, the more internal telomeric sequences are seen (Meyne et al. 1990). Interestingly, no interstitial TTAGGG sequences were found in the oyster, Crassostrea gigas (n = 10), a more primitive bivalve (Guo and Allen 1997), or in C. angulata (Cross et al. 2005), whereas the current study (Fig. 4) was able to demonstrate their presence in the more recent A. irradians (1.5 million years old) and only slightly in the deep-sea scallop, P. magellanicus (65 million years old). Zou et al. (2002) found in a species of deer, internal telomere sequences (subtelomeric) are also sites of fragility that may be leftover remnants of Robertsonian fusion that may actually contribute to further chromosome instability. In humans, approximately 50% of all subtelomeric sequences were generated after the chimpanzee/human divergence (Linardopoulou et al. 2005).

A. irradians is often labeled semelparous (Barber & Blake 1986, Bricelj et al. 1987a, Estabrooks 1999, Tettelbach et al. 1999 among others), but there appears to be no selective advantage to the bay scallop having such a short life span. A semelparous existence is usually found when there is a need to put all of one's energy into a single reproductive effort with death resulting shortly thereafter. This is seen, for example, in the Pacific salmon, Oncorhynchus spp., in which all of the energy needed for the reproductive process is brought in from the ocean in the form of stored nutrients to be used upstream (Morbey et al. 2005). To the contrary, the bay scallop enters into its second year post spawning by doubling in weight from September to the end of November (Bricelj et al. 1987b, Tettelbach et al. 2002). Lipids, carbohydrates, and protein are stored for the winter, and many may survive to at least initiate gametogenesis in the early spring, though most rarely survive to complete a second spawning (Belding 1910). Bricelj & Krause (1992) found a high rate of survival to a second reproductive season without completion (90% surviving until March). A. irradians appears to be caught by surprise, so to speak, let down by telomeric deletions, perhaps better labeled as a case of "iteroparous interruptus."

Darwinian theory would infer that this single life history trait (reproduction) has been optimized over time, but a more realistic view may suggest that the physiological constraints of living under species-specific opportunities might yield a strategy that, whereas not optimal, is still "good enough" (Darlington 1977, Tuomi et al. 1983). It is postulated that at least part of the key chromosomal material lost over time were telomeres. The energy-producing digestive gland with its rapidly dividing cells appears to be the key organ to lose sufficient telomeres to initiate senescence and the subsequent death of the bay scallop. In comparing the telomeres of the digestive gland of a two-year-old Argopecten purpuratus with those of the first and second year Argopecten irradians, it can be seen that A. purpuratus has longer telomeres (Fig. 1). Semiquantitative estimates show that a two-year-old A. purpuratus contains telomere restriction fragment (TRF) lengths that are much longer than those of a first year A. irradians. Figure 3 shows even shorter telomeres in the digestive gland of A. irradians entering its third year. The telomeres of the heart and adductor muscle do not shorten as these tissues do not generally undergo cell division (Fig. 3,4).

The gill and kidney also demonstrates telomere losses as would be expected, but not to the extent seen in the digestive gland (data not shown). This might be explained in the kidney, as this organ stores numerous excretion granules that are laid down layer by layer over an extended period to be finally expelled in the urinary tract (George et al. 1980, Morse 1987). This process would result in a slower cellular turnover and fewer lost telomeres.

Whereas the final time of death may be caused by variations in the environment coupled with the condition of individual scallops as suggested by Bricelj et al. (1987b), it is most likely that telomere loss is the coarse adjustment of the mitotic clock that determines the average life span of this species. The fine tuning may be modulated by several factors such as the initial number of telomeres, and the individual rate of cell turnover. Scallops in deeper and colder water may have a slower rate of metabolism, whereas those spawned later in the season will face different conditions at different points in their lives. Those that store fewer nutrients for the second upcoming winter may be in a weaker condition, as would be the case of those infected with parasites.

This could also account for the observations that senescence in scallops may be size related as well as age related (Gutsell 1930, Orensantz 1986, Bricelj et al. 1987b) with smaller scallops surviving longer for having had fewer cellular replications and thus fewer telomeres lost.

Bay scallop mortality can take place over a period of up to several months, usually beginning in early winter and running through to spring in the Northeast (Belding 1910, Bricelj et al. 1987b). It is believed that other theories of aging, including the accumulation of genetic errors over time, the damage caused by free radicals or an excessive metabolic rate may have their effects on defining the date of death of individual scallops, but not on the overall life span of the species.

It is proposed that A. irradians has the potential of a longer life, and only the unfortunate loss of key chromosomal material from an original tetraploid relict prevents it from doing so. A consequence of evolving into a species that has adapted to shallow bays may have been the loss of key telomeric sequences. It seems to make all of the preparations necessary for survival through the upcoming winter and beyond, but rarely lives to complete a second reproductive season. Genetic manipulation to increase the number of telomeres in A. irradians could possibly grant the bay scallop several additional reproductive opportunities that could help ameliorate the impact of some larval catastrophes in which there were no concomitant catastrophic losses of the spawner population.

This leads to the question that if an extended life span could be the answer to some of the problems facing A. irradians, then how successful is A. purpuratus in Chile? The industry there faces a different set of problems maintaining a successful threshold level of scallops in the wild, mainly that of illegal extraction. All year cohorts are considered seed and are taken indiscriminately by divers (Stotz & Gonzalez 1997), leaving aquaculture to account for the majority of scallop production today in Chile (von Brand et al. 2006). Genetic crosses of A. irradians irradians and A. purpuratus may have the possibility of extending the life span of the former. Both are cold water species with n = 16 and identical karyotypes, 5st (subtelocentric) and 11t (telocentric) (Gajardo et al. 2002, Wang & Guo 2004). Waller (1969), states that A. purpuratus, which is 4.5-5 million years older than A. irradians, has undergone little change over time. In addition, Chen et al. (1991) found 90% of crosses between Chlamys farreri (n = 19) and A. irradians (n = 16), survived up to 12 days. It seems reasonable to expect that a cross between A. irradians and A. purpuratus would fare better. The El Nino event of 1983, when ambient water temperatures dramatically increased, resulted in a sixty-fold increase in the normal scallop population. Wolff (1987), has suggested that A. purpuratus, a normally cold-water species, may have retained many of its warm water characteristics from the tropical/subtropical Miocene. If this true, a cross between A. purpuratus and A. irradians concentricus (Say) or A. irradians amplicostatus (Dahl), may yield similar results. Currently, research is underway to determine the viability of crosses between A. irradians irradians and A. purpuratus and to measure the resulting telomeres, if such crosses are successful.

ACKNOWLEDGMENTS

The author thanks Dr. Sarah Oktay of the University of Massachusetts Field Station and Valerie Hall for their critical input. This research was supported by grants from the Nantucket Marine and Coastal Resources Department and the PADI Foundation.

LITERATURE CITED

Alarcon, E. & M. Wolff. 1991. Estudio biologico pesquero sobre el recurso de ostiones (Argopecten purpuratus) de bahia Tongoy durante el fenomeno El Nino 1982-83. Invest. Pesq. 32:167-173.

Barber, B. J. & N. J. Blake. 1986. Reproductive effort and cost in the bay scallop, Argopecten irradians concentricus. Int'l. J. of Invert. Reprod. and Develop. 10:51-57.

Beaumont, A. R. & E. Zouros. 1991. Genetics of scallops. In: S.E. Shumway, editor. Scallops: biology, ecology and aquaculture. Developments in aquaculture and fisheries science. Vol. 21. Amsterdam: Elsevier. pp. 585-623.

Belding, D. L. 1910. A report upon the scallop fishery of Massachusetts. The Commonwealth of Massachusetts. Boston. 150 pp.

Blackburn, E. H. 1991. Structure and function of telomeres. Nature 350:569-573.

Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C.-P. Chiu, G. B. Morin, C. B. Harley, J, W. Shay, S. Lichtsteiner & W. E. Wright. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279:349-352.

Bricelj, V. M., J. Epp & R. E. Malouf. 1987a. Comparative physiology of young and old cohorts of bay scallop Argopecten irradians irradians (Lamarck): mortality, growth and oxygen consumption. J. Exp. Mar. Biol. Ecol. 112:73-91.

Bricelj, V. M., J. Epp & R. E. Malouf. 1987b. Intraspecific variation in reproductive and somatic growth cycles of bay scallops. Argopecten irradians. Mar. Ecol. Prog. Ser. 36:123-137.

Bricelj, V. M. & M. K. Krause. 1992. Resource allocation and population genetics of the bay scallop, Argopecten irradians irradians: effects of age and allozyme heterozygosity on reproductive output. Mar. Biol. 113:253-261.

Carlson, J. C. & J. C. M. Riley. 1998. A consideration of some notable aging theories. Exp. Gerontol. (1/2):127-134.

Chen, Q., J. Xiang, Y. Qin, B. Kou & H. Wang. 1991. Ref. in: Wang, Y. & X. Guo. 2004. Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution. Biol. Bull. 207:247-256.

Cross, I., E. Diaz. I. Sanchez & L. Rebordinos. 2005. Molecular and cytogenetic characterization of Crassostrea angulata chromosomes. Aquaculture 247:135-144.

Darlington, P. J., Jr. 1977. The cost of evolution and the imprecision of adaptation. Proc. Natl. Acad. Sci. USA 74:1647-1651.

DiSalvo, L. H., E. Alarcon, E. Martinez & E. Uribe. 1984. Progress in mass culture of Chlamys (Argopecten) purpuratus Lamarck (1819) with notes on its natural history. Rev. Chil. Hist. Nat. 57:34-45.

Epp, J., V. M. Bricelj & R. E. Malouf. 1988. Seasonal partitioning and utilization of energy reserves in two age classes of the bay scallop Argopecten irradians irradians (Lamarck). J. Exp. Mar. Biol. Ecol. 121:113-136.

Estabrooks, S. L. 1999. The telomeres of the bay scallop, Argopecten irradians irradians (Lamarck). J. Shellfish Res. 18:401-404.

Gajardo, G., M. Parragez & N. Colihueque. 2002. Karyotype analysis and chromosome banding of the Chilean-Peruvian scallop, Argopecten purpuratus (Lamarck, 1819). J. Shellfish Res. 21:585-590.

George, S. G., B. J. S. Pirie & T. L. Coombs. 1980. Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.). J. Exp. Mar. Biol. Ecol. 42:143-156.

Guo, X. & S. K. Allen, Jr. 1997. Fluorescence in situ hybridization of vertebrate telomere sequence to chromosome ends of the Pacific oyster, Crassostrea gigas Thunberg. J. Shellfish Res. 16: 87-89.

Gutsell, J. S. 1930. Natural history of the bay scallop. Bull. U.S. Bur. Fish. 45:569-632. Harley, C.B. 1991. Telomere loss: mitotic clock or genetic time bomb? Murat. Res. 256:271-282.

Harley, C. B. 1991. Telomere loss: mitotic clock or genetic time bomb? Mutat. Res. 256:271-282.

Harley, C. B., H. Vaziri, C. M. Counter & R. C. Allsopp. 1992. The telomere hypothesis of cellular ageing. Exp. Gerontol. 27:375-382.

Harman, D. 1956. Aging-A theory based on free radical and radiation chemistry. J. Gerontol. 12:298-300.

Haussmann, M. F., D. W. Winkler & C. M. Vieck. 2005. Longer telomeres associated with higher survival in birds. Biol. Lett. 1:212-214.

Herbert, B. S., A. E. Pitts, S. I. Baker, S. E. Hamilton, W. E. Wright, J. W. Shay & D. R. Corey. 1999. Inhibition of human telomerase in immortal human ceils leads to progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. USA 96:14276-14281.

Joeng, K. S., E. J. Song, K.-J. Lee & J. Lee. 2004. Long lifespan in worms with long telomeric DNA. Nat. Genet. 36:607-611.

Klapper, W., K. Heidorn, K. Kuhne, R. Parwaresch & G. Krupp. 1998a. Telomerase activity in 'immortal' fish. FEBS Lett. 434: 409-412.

Klapper, W., K. Kuhne, K. K. Singh, K. Heidorn, R. Parwaresch & G. Krupp. 1998b. Longevity of lobsters is linked to ubiquitous telomerase expression. FEBS Lett. 439:143-146.

Koziol, C., R. Borojevic, R. Steffen & W. E. G. Muller. 1998. Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells. Mech. Age. Dev. 100:107-120.

Kulju. K. S. & J. M. Lehman. 1995. Increased p53 protein associated with aging in human diploid fibroblasts. Exp. Cell Res. 217:336-345.

Levine, A. J., A. Chang & D. Dittmer. 1993. The p53 tumor suppressor gene. J. Lab. Clin. Med. 123:817-823.

Levy, M. Z., R. C. Allsopp, A. B. Futcher. C. W. Greider & C. B. Harley. 1992. Telomere end-replication problem and cell aging. J. Mol. Biol. 225:951-960.

Linardopoulou, E. V., E. M. Williams, Y. Fan, C. Friedman, J. M. Young & B. J. Trask. 2005. Human subtelomeres are hot spots of interchromosomal recombination and segmental duplication. Nature 437:94-100.

Meyne, J., R. J. Baker, H. H. Hobart, T. C. Hsu, O. A. Ryder, O. G. Ward, J. E. Wiley, D. H. Wurster-Hill T. L. Yates & R. K. Moyzis. 1990. Distribution of non-telomeric sites of the [(TTAGGG).sub.n] telomeric sequence in vertebrate chromosomes. Chromosoma 99: 3-10.

Meyne, R. K., R. L. Ratliffe & R. K. Moyzis. 1989. Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc. Natl. Acad. Sci. USA 86:7049-7053.

Morbey, Y. E., C. E. Brassil & A. P. Hendry. 2005. Rapid senescence in Pacific salmon. The Amer. Naturalist 166:556-568.

Morse, M. P. 1987. Comparative functional morphology of the bivalve excretory system. Am. Zool. 27:737-746.

Orensantz, J. M. 1986. Size, environment, and density: the regulation of a scallop stock and its management implications. Can. Spec. Publ. fish. Aquat. Sci. 92:195-227.

Plohl, M., E. Prats, A. Martinez-Lage, A. Gonzalez-Tizon, J. Mendez & L. Cornudella. 2002. Telomeric localization of the vertebrate-type hexamer repeat, [(TTAGGG).sub.n], in the wedgeshell clam Donax trunculus and other marine invertebrate genomes. J. Biol. Chem. 277:19839-19846.

Rocco, L., D. Costagliola & V. Stingo. 2001. [(TTAGGG).sub.n] telomeric sequence in selachian chromosomes. Heredity 87:583-588.

Ropes, J. W. 1985. Modern methods used to age oceanic bivalves. Nautilus 99:53-57.

Rose, M. R. 1991. Evolutionary biology of aging. New York: Oxford University Press.

Shay, J. W. & W. E. Wright. 2000. Hayflick. His limit, and cellular ageing. Nat. Rev. Mol. Cell Biol. 1:72-76.

Sokolov, E. P. 2000. An improved method for DNA isolation from mucopolysaccharide-rich mollluscan tissues. J. Moll. Stud. 66:573-575.

Stotz, W. B. & S. A. Gonzalez. 1997. Abundance, growth and production of the sea scallop Argopecten purpuratus (Lamarck 1819): bases for sustainable exploitation of natural scallop beds in north-central Chile. Fish. Res. 32:173-183.

Tettelbach, S. T., C. F. Smith, R. Smolowitz, K. Tetrault & S. Dumais. 1999. Evidence for fall spawning of northern bay scallops Argopecten irradians irradians (Lamarck 1819) in New York. J. Shellfish Res. 18:47-58.

Tettelbach, S. K., C. F. Smith, P. Wenczel & E. Decort. 2002. Reproduction of hatchery- reared and transplanted wild bay scallops, Argopecten irradians irradians, relative to natural populations. Aquacult. Internat. 10:279-296.

Thompson, I., D. S. Jones & D. Dreibelbis. 1980. Annual internal growth banding and life history of the ocean quahog Artica islandica (Mollusca:Bivalvia). Mar. Biol. 57:25-34.

Tuomi, J., T. Hakala & E. Haukioja. 1983. Alternative concepts of reproductive effort, costs of reproduction, and selection in life-history evolution. Am. Zool. 23:25-34.

Turekian, K. K., J. K. Cochran, D. P. Kharkar, R. M. Cerrato, J. R. Vaisnys, H. L. Sanders, J. F. Grassle & J. A. Allen. 1975. Slow growth rate of a deep-sea clam determined by [sup.228]Ra chronology. Proc. Natl. Acad. Sci. USA 72:2829-2832.

Vaziri, H. & S. Benchimol. 1996. From telomere loss to p53 induction and activation of a DNA-damage pathway at senescence: the telomere loss/DNA damage model of cell aging. Exp. Gerontol. 31:295-301.

Vaziri, H. & S. Benchimol. 1998. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr. Biol. 8:279-282.

Vitturi, R., A. Libertini, L. Sineo, I. Sparacio, A. Lannino, A. Gregorini & M. Colomba. 2005. Cytogenetics of the land snails Cantareus aspersus and C. mazzullii (Mollusca:Gastropoda:Pulmonata). Mieron 36:351-357.

von Brand, E., G. E. Merino, A. Abarca & W. Stolz. 2006. Scallops: biology, ecology and aquaculture. In: S.E. Shumway & G.J. Parsons, editors. Amsterdam: Elsevier B.V 1293-1314.

Wada, K. 1978. Chromosome karyotypes of three bivalves: the oysters, Isognomon alatus and Pinctada imbricata, and the bay scallop, Argopecten irradians irradians. Biol. Bull. 155:235-245.

Waller, T. R. 1969. The evolution of the Argopecten gibbus stock (Mollusca:Bivalvia), with emphasis on the Tertiary and Quaternary species of eastern North America. J. Paleon. 4, part II of II, supplement No. 5.

Wang, Y. & X. Guo. 2004. Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution. Biol. Bull. 207:247-256.

Whikehart, D. R., S. J. Register, Q. Chang & B. Montgomery. 2000. Relationship of telomeres and p53 in aging bovine corneal endothelial cell cultures. Invest. Ophthal. Visual Sci. 41:1070-1075.

Wolff, M. 1987. Population dynamics of the Peruvian scallop Argopecten purpuratus during the El Nino phenomenon of 1983. Can. J. Fish. Aquat. Sci. 44:1684-1691.

Wright, W. E. & J. W. Shay. 2005. Telomere biology in aging and cancer. J. Am. Ger. Soc. 53:S292-S294.

Zou, Y., X. Yi, W. E. Wright & J. W. Shay. 2002. Human telomerase can immortalize Indian Muntjac cells. Exp. Cell Res. 281: 63-76.

STEPHEN L. ESTABROOKS * Nantucket Marine Laboratory, 0 Easton Street, Nantucket, Massachusetts 02554

* E-mail: [e-mail]